DNA-PKcs controls calcineurin mediated IL-2 production in T lymphocytes

Abstract

Loss of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity in mammals results in severe combined immuno-deficiency (SCID). This SCID phenotype has been postulated to be due solely to the function of DNA-PKcs in V(D)J recombination, a process critical for lymphocyte maturation. However; we show that DNA-PKcs is required for IL-2 production via regulation of the calcineurin signaling pathway. Reducing DNA-PKcs activity in activated T cells either by shRNA or an inhibitor significantly reduced IL-2 production by blocking calcineurin activity and the translocation of NFAT into the nucleus. Additionally, we show that DNA-PKcs exerts its effect on calcineurin by altering the expression of the endogenous calcineurin inhibitor Cabin1 through activation of the kinase CHK2, a known Cabin1 regulator. The discovery of DNA-PKcs as a potent regulator of IL-2 production will drive continued investigation of small molecule inhibition of this enzyme within the clinic.

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

“…34 Additionally, our laboratory determined that DNA-PKcs controls NFAT-mediated transcription through indirect regulation of proteasomal degradation of the calcineurin inhibitor Cabin1. 6 Interestingly, in the report by Jain et al, they suggest that phosphorylation of Egr1 by CKII regulates its activity by promoting Egr1's interaction with and subsequent degradation by the proteasome. 32 It is possible that phosphorylation by DNA-PKcs at this site that impedes, possibly through a conformational change, proteasomal interaction preventing degradation.…”

“…5 Interestingly, this enzyme is robustly expressed in mature lymphocytes and consistently activated by various lymphocyte stimulants. 6,7 This emphasizes a function for DNA-PKcs in the mature immune system that is yet to be clearly defined.…”

“…Inhibition of DNA-PKcs blocked calcineurin activity thereby preventing the translocation of NFAT to the nucleus and expression of cytokine IL2. 15 Herein, we report that DNA-PKcs also regulates expression of the immediate early response gene (IEG) Egr1 (early growth response 1), a transcription factor critical for cytokine production. [16][17][18] IEG genes such as Egr1 are transcribed within minutes of TCR stimulation to rapidly turn on transcription of genes needed for immune cell function.…”

“…These defects have been primarily attributed to the function of DNA-PKcs in V(D)J recombination, which is required for antibody and receptor diversity in adaptive immune cells ( 5 ). Interestingly, this enzyme is robustly expressed in mature lymphocytes and consistently activated by various lymphocyte stimulants ( 6 , 7 ). This emphasizes a function for DNA-PKcs in the mature immune system that is yet to be clearly defined.…”

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

“…32 Our laboratory determined that DNA-PKcs controls NFAT-mediated transcription through indirect regulation of proteasomal degradation of the calcineurin inhibitor Cabin1. 6 In contrast, DNA-PKcs has also been shown to promote ubiquitination of proteins for proteasome targeting. For instance, in order to arrest transcription at DSB sites, Caron et al discovered that DNA-PKcs promotes ubiquitination of RNA polymerase II by HECT E3 ubiquitin ligase WWP2 thereby thwarting transcription.…”